| Objective: Both bone marrow derived mesenchymal stem cells(BM-MSCs)and adipose derived mesenchymal stem cells(AD-MSCs)play an importmant role in injured tissue repairment,which is main through secreting exosomes.However,it is unknown the advantages and disadvantages of BM-MSCs and AD-MSCs for repairment of ischemic cardiomyocytes.In this study,we will explore the cells characteristics,exosomes secrection and cardioprotection of BM-MSCs and AD-MSCs derived from C57BL/6 mice.Method: 6-10 weeks old male C57BL/6 mice were used to isolate AD-MSCs and BM-MSCs.Cell proliferation and double times were measured by MTS assay and cell counting respectively.Flow cytometry was applied to measure the MSCs markers(CD29,Sca-1 and CD34)and autophagy.The conditioned medium was acquired after the passage 4 of AD-MSCs and BM-MSCs cultured with serum-free medium for 48 hours,and exosomes were isolated from AD-MSCs(Exo-AD)and exosomes isolated from BM-MSCs(Exo-BM)were isolated from the conditioned medium by ultrafiltration centrifugation and the Exo Quick-TC kit.The exosomes average size and distribution were observed by Nanosight.The exosomes and other proteins concentration was quantitated by DC protein assay.Exosomes marker CD63 and autophagy related molecule Beclin-1 were measured by Western blot.The neonatal cardiomyocytes were isolated from 1-3 days old SD rats following the cardiomyocytes isolation kit protocol.The cardiomyoctes ischemic model was built according to the results of MTS and LDH assay.And the uptake of Exo-AD and Exo-BM was evaluated through using exosomes labeled PKH26 and incubating with cardiomyocytes for 12 hours,then observed the red labeled exosomes inside cardiomyocytes under fluorescence microscope.Cardiomyocytes were respective treated with Exo-AD and Exo-BM for 72 h hypoxia,MTS assay was used to measure the cells survival.The mitochondrial membrane potential(ΔΨm)of Cardiomyocytes was tested by using JC-1 assay.Also,mi RNAs sequence assay(mi R-seq)was applied to measure the mi RNAs in both Exo-AD and Exo-BM.Results: The experiment found that AD-MSCs have a faster proliferation and a better ability of exosomes secretion than BM-MSCs.In contrary,the autophagy expression was higher in BM-MSCs.The ischemic model was built successfully.Both Exo-AD and Exo-BM could be uptook by cardiomyocytes.Compare with the control group,both Exo-AD and Exo-BM have the function of cardioprotection,but the function of Exo-BM was better than that of Exo-AD when using the same quantity exosomes.mi R-seq result showed that about 243 mi RNAs were different in Exo-AD and Exo-BM,including mi R-21,mi R-30,mi R-221 and mi R-223,which are associated with cardioprotection.Conclusion: This study demonstrated that there were differences in cell characteristics,exosomes secrection and cardioprotection of BM-MSCs and AD-MSCs.And the main reason may be associated with the differences in mi RNAs contained in Exo-AD and Exo-BM,which provide a theory evidence for clinical selection of MSCs. |
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